Endotoxin is a lipopolysaccharide ("LPS") found in the outer membrane of most gram-negative bacteria. Endotoxin's biologically active moiety, lipid A, associates with a LPS-binding protein ("LBP") in mammals; the resultant LPS/LBP complex binds to the CD14 surface receptor on macrophages and other LPS-responsive cells (Kielian and Blecha, Immunology 29: 187 (1995)). Exposure to endotoxin can thereby lead to fever and leukopenia followed by leukocytis, and in larger doses, shock. Accordingly, detection and quantification of endotoxin is an important concern in the manufacture of pharmaceutical formulations. More than 20 assays for the detection of endotoxin have been reported, but an assay based on the coagulation of Limulus polyphemus blood is the method of choice (Hurley, Clin. Microbiol. Rev. 8: 268 (1995). It has the advantages of increased sensitivity, potential for quantitation, reactivity with the biologically active component, lipid A and relative convenience of operation. It makes use of endotoxin-induced activation of enzymes in the Limulus coagulation cascade; these enzymes then react with a clottable protein, forming a detectable gel that is indicative of the presence of endotoxin (the "LAL assay"; Sullivan et al., in: Mechanisms in Bacterial Toxicology (Bernheiner, ed.), John Wiley & Sons, NY (1976), p. 217). The LAL assay is capable of detecting endotoxin at levels as low as several picograms of endotoxin per ml of sample.
However, incorporation of endotoxin into lipid bilayers, liposomes or lipid complexes masks the lipid A moiety from LBP and assay enzymes, inhibiting the ability of assay enzymes to interact with lipid A and thereby decreasing the sensitivity of the assay (J. Dijkstra et al., J. Immunol. 138: 2663 (1987)). Therefore, accurate assays for the detection of endotoxin sequestered in such structures must provide a means for unmasking endotoxin's lipid A moiety. Previously known methods have proved to be inappropriate, ineffective or too inefficient to have practical use. For example, Gutierrez and Liebana, (Ann. Biol. Clin. 51: 83 (1993)) noted that immunological methods are expensive and insensitive. The method developed by Trubetskoy et al. (FEBS Lett. 269; 79 (1990)), based on the development of a fluorescent derivative of LPS, provided important information for understanding LPS-lipid bilayer interaction, but was unsuitable for detecting unlabeled LPS. Schmidtgen and Brandl (Journal of Liposome Research 5;109 (1995)) describe a method based on solubilization of liposomes with ethanol followed by a series of labor intensive ultrafiltration steps. This method is time consuming and expensive. An efficient method having a minimum number of steps and avoiding exposure of the sample to potentially contaminated glassware and solvents is needed.
This invention provides a method which is able to reliably, and with sufficient sensitivity, detect the presence of endotoxin in lipid bilayers, liposomes or lipid complexes. Detergents disturb the stability of lipid bilayers, liposomes and lipid complexes, thus solubilizing the structures. When present in sufficient concentrations in suspensions of lipid bilayers, liposomes or lipid complexes, detergents disrupt their structures, forming micelles therefrom which contain the detergent, lipid and lipid-soluble lipid contents of the bilayer, liposome or complex. Detergents thus solubilize the lipid bilayers, liposomes or lipid complexes. Endotoxin assays require that detergents be used at a concentration sufficient for the detergent to solubilize substantially all of the lipid bilayers, liposomes or lipid complexes present in a suspension, so that substantially all of the endotoxin present in these structures is released therefrom for detection and quantification. Solubilization of less than substantially all of the available lipid bilayers, liposomes or lipid complexes does not release substantially all of the endotoxin present, and does not lead to a reliable result when an endotoxin assay is applied to the resulting micellar suspension.
However, some detergents, when present in sufficient concentration to solubilize lipid bilayers, liposomes or lipid complexes, can also affect molecules enmeshed in these lipid-based structures other than the amphipathic lipids which are the structures' primary constituents. The structure of lipid A, for example, is affected by detergents which have previously been applied in endotoxin assays such that the assay detection means have been inhibited from interacting with the endotoxin moiety. High concentrations of such detergents thus may decrease the sensitivity of the endotoxin assays; at sufficiently high detergent concentrations, assay sensitivity is affected to a degree such that the results are not reliable.
Detergents suitable for use in the endotoxin assays of the present invention must thus be able to solubilize substantially all of the lipid bilayers, liposomes or lipid complexes present in a suspension at a detergent concentration at which detergent-mediated inhibition of the assay detection means is minimized. This invention provides an assay for detecting the presence of endotoxin in lipid bilayers, liposomes or lipid complexes, in which the detergents used can solubilize the bilayers, liposomes or complexes so as to give micellar suspensions in which the iipid concentration is at least about 1.75 mg/ml. At the same time, the detergents inhibit activity of the assay detection means by a factor of less than about 100.
No detergents previously applied to endotoxin assays meet these criteria. For example, Pyrosperse.TM., which is the detergent made available in connection with the LAL assay currently applied to liposomes, cannot solubilize the liposomes at the levels achieved by the detergents used in the method of this invention.